Abstract

The PTPN11 (protein-tyrosine phosphatase, non-receptor type 11) gene encodes SHP2, a cytoplasmic PTP that is essential for vertebrate development. Mutations in PTPN11 are associated with Noonan and LEOPARD syndrome. Human patients with these autosomal dominant disorders display various symptoms, including short stature, craniofacial defects and heart abnormalities. We have used the zebrafish as a model to investigate the role of Shp2 in embryonic development. The zebrafish genome encodes two ptpn11 genes, ptpn11a and ptpn11b. Here, we report that ptpn11a is expressed constitutively and ptpn11b expression is strongly upregulated during development. In addition, the products of both ptpn11 genes, Shp2a and Shp2b, are functional. Target-selected inactivation of ptpn11a and ptpn11b revealed that double homozygous mutants are embryonic lethal at 5-6 days post fertilization (dpf). Ptpn11a-/-ptpn11b-/- embryos showed pleiotropic defects from 4 dpf onwards, including reduced body axis extension and craniofacial defects, which was accompanied by low levels of phosphorylated Erk at 5 dpf. Interestingly, defects in homozygous ptpn11a-/- mutants overlapped with defects in the double mutants albeit they were milder, whereas ptpn11b-/- single mutants did not show detectable developmental defects and were viable and fertile. Ptpn11a-/-ptpn11b-/- mutants were rescued by expression of exogenous ptpn11a and ptpn11b alike, indicating functional redundance of Shp2a and Shp2b. The ptpn11 mutants provide a good basis for further unravelling of the function of Shp2 in vertebrate development.

Expression of zebrafish ptpn11a and ptpn11b mRNA during embryogenesis.

(a) RNA was isolated from zebrafish embryos at 4 hpf, 10 hpf, 1 dpf, 2 dpf, 3 dpf and 5 dpf. Reverse transcription PCR was performed to detect the indicated genes; reverse transcriptase was omitted from the reaction as a control (-RT). (b) Quantitative PCR for ptpn11a and ptpn11b gene expression was performed from a pool of wild-type embryos (n = 30) collected at 10 hpf, 1 dpf, 2 dpf, 3 dpf and 5 dpf. The values were normalized using Actin as a control and relative expression is depicted here (value at 5 dpf set to 1.0). Error bars indicate standard error of the mean. (c) Whole-mount in situ hybridization using ptpn11a and ptpn11b specific probes of 4 hpf, 10 hpf, 30 hpf, 2 dpf, 3 dpf and 5 dpf embryos. Lateral and dorsal views are given as indicated.

(a) PTP activity of pGEX, WT-Shp2a, Shp2a-D61G, WT-Shp2b and Shp2b-D61G was assayed using p-nitrophenylphosphate (pNPP) and quantified spectrophotometrically. Approximately 1 µg purified fusion protein was used in the assay. Experiments were done in quadruplicate; averages are depicted and error bars indicate standard error of the mean. (b) Equivalent amounts of fusion protein that were used in the activity assays in (a) were run on an SDS-PAGE gel and stained with Coomassie blue.

Sequence analysis of homozygous ptpn11a and ptpn11b mutants. (a) A C to T mutation in ptpn11ahu3459 resulted in a glutamine to a STOP mutation. (b) A T to A mutation in ptpn11bhu5920 changed lysine to a STOP mutation. (c) Schematic representation of the exon organization and structural domains of ptpn11a and ptpn11b. Nonsense mutations upstream of the catalytic domain are represented by red arrows.

Morphology of 5(a) Representative wild-type, (b) ptpn11a-/-, (c) ptpn11b-/- and (d) double homozygous mutant are depicted. (e) The length was measured from nose to tip of tail at the same magnification. Bars show average length of the studied genotypes (n = 10-20). Statistics were determined using a student's t-test; * indicates a p value <0.05.

WT, ptpn11a-/-, ptpn11b-/- and ptpn11a-/- ptpn11b-/- embryos were lyzed and immunoblotted with phosho-specific antibody for ERK and AKT; membranes were then reprobed for total Erk and Akt expression to control for loading. The blots were quantified and the ratio of phosphorylated protein/total protein from three independent experiments is indicated below each lane.